Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence

Ayinuola, Yetunde A.; Brito-Robinson, Teresa; Ayinuola, Olawole; Beck, Julia; Cruz‐Topete, Diana; Lee, Shaun W.; Ploplis, Victoria A.

doi:10.1074/jbc.ra120.016262

Cited by 7 publications

(12 citation statements)

References 56 publications

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“…The affinity binding study was performed at 25°C in BIAcoreX100 (GE Healthcare) system with 10 mM Na + -Hepes/150 mM NaCl/3 mM EDTA/0.05% polysorbate 20, pH 7.4 (HBS-EP+). The chip activation, immobilization, and blocking were accomplished as previously described ( Ayinuola et al, 2020 ). The binding experiments were done in triplicate using hPg as analyte at concentrations ranging from 9 to 2,400 nM.…”

Section: Methodsmentioning

confidence: 99%

Relationships Between Plasminogen-Binding M-Protein and Surface Enolase for Human Plasminogen Acquisition and Activation in Streptococcus pyogenes

et al. 2022

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The proteolytic activity of human plasmin (hPm) is utilized by various cells to provide a surface protease that increases the potential of cells to migrate and disseminate. Skin-trophic Pattern D strains of Streptococcus pyogenes (GAS), e.g., GAS isolate AP53, contain a surface M-protein (PAM) that directly and strongly interacts (Kd ~ 1 nM) with human host plasminogen (hPg), after which it is activated to hPm by a specific coinherited bacterial activator, streptokinase (SK2b), or by host activators. Another ubiquitous class of hPg binding proteins on GAS cells includes “moonlighting” proteins, such as the glycolytic enzyme, enolase (Sen). However, the importance of Sen in hPg acquisition, especially when PAM is present, has not been fully developed. Sen forms a complex with hPg on different surfaces, but not in solution. Isogenic AP53 cells with a targeted deletion of PAM do not bind hPg, but the surface expression of Sen is also greatly diminished upon deletion of the PAM gene, thus confounding this approach for defining the role of Sen. However, cells with point deletions in PAM that negate hPg binding, but fully express PAM and Sen, show that hPg binds weakly to Sen on GAS cells. Despite this, Sen does not stimulate hPg activation by SK2b, but does stimulate tissue-type plasminogen activator-catalyzed activation of hPg. These data demonstrate that PAM plays the dominant role as a functional hPg receptor in GAS cells that also contain surface enolase.

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Section: Methodsmentioning

confidence: 99%

Relationships Between Plasminogen-Binding M-Protein and Surface Enolase for Human Plasminogen Acquisition and Activation in Streptococcus pyogenes

et al. 2022

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“…In the case of hPg [D 139,413,518 N], the digested fragment was ligated into hPg [D 139 N]-pMT-PURO. The construction of the plasmid coding for hPg [D 219 N] was previously reported ( Ayinuola et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

“…We have demonstrated, with the aid of through-space lysine isosteres of PAM ( Rios-Steiner et al, 2001 ; Yuan et al, 2019 ; Qiu et al, 2020 ), that the acquisition of hPg and its conversion to hPm by the endogenous GAS hPg activator, streptokinase (SK), assists GAS in the degradation of barriers to its dissemination ( Vu et al, 2021 ). Importantly, we showed that PAM binding to K2 hPg displaces the intramolecular interactions maintained by this domain, giving rise to a conformational switch that expedites streptokinase-mediated activation of hPg ( Bhattacharya et al, 2014 ; Ayinuola et al, 2021 ). Likewise, the replacement of Asp 219 of the anionic center of K2 hPg LBS, ( Ayinuola et al, 2021 ) while abolishing PAM-binding, produces a hPg with a relaxed conformation having a rapid and optimal activation potential ( Ayinuola et al, 2021 ).…”

Section: Introductionmentioning

confidence: 95%

“…Importantly, we showed that PAM binding to K2 hPg displaces the intramolecular interactions maintained by this domain, giving rise to a conformational switch that expedites streptokinase-mediated activation of hPg ( Bhattacharya et al, 2014 ; Ayinuola et al, 2021 ). Likewise, the replacement of Asp 219 of the anionic center of K2 hPg LBS, ( Ayinuola et al, 2021 ) while abolishing PAM-binding, produces a hPg with a relaxed conformation having a rapid and optimal activation potential ( Ayinuola et al, 2021 ).…”

Section: Introductionmentioning

confidence: 95%

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Inactivation of the lysine binding sites of human plasminogen (hPg) reveals novel structural requirements for the tight hPg conformation, M-protein binding, and rapid activation

Ayinuola¹

2023

Front. Mol. Biosci.

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Accelerated activation of the human plasminogen zymogen (hPg) to two-chain active plasmin (hPm) is achieved following conformational changes induced by ligand-binding at the lysine-binding sites (LBSs) in four of the five hPg kringle domains. In this manner, pattern D skin-trophic strains of Group A streptococci (GAS), through the expression of surface plasminogen-binding M-protein (PAM), immobilize surface hPg, thereby enabling rapid hPg activation by GAS-secreted streptokinase (SK). Consequently, GAS enhances virulence by digesting extracellular and tight cellular junctional barriers using hPm activity. Many studies have demonstrated the singular importance of the kringle-2 domain of hPg (K2hPg) to PAM-binding using hPg fragments. Recently, we showed, using full-length hPg, that K2hPg is critical for PAM binding. However, these studies did not eliminate any modulatory effects of the non-K2hPg LBS on this interaction. Moreover, we sought to establish the significance of the intramolecular interaction between Asp219 of the LBS of K2hPg and its serine protease domain binding partner, Lys708, to conformational changes in hPg. In the current study, selective inactivation of the LBS of K1hPg, K4hPg, and K5hPg revealed that the LBS of these kringle domains are dispensable for hPg binding to PAM. However, the attendant conformational change upon inactivation of K4hPg LBS increased the affinity of hPg for PAM by an order of magnitude. This finding suggests that the native hPg conformation encloses PAM-binding exosites or sterically hinders access to K2hPg. While simultaneous inactivation of the LBS of K1hPg, K4hPg, and K5hPg inhibited hPg/SK association alongside hPg activation, the replacement of Lys708 generated a slight conformational change that optimally accelerated hPg activation. Thus, we accentuate disparate functions of hPg LBS and conclude, using intact proteins, that K2hPg plays a central role in regulating hPg activation.

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“…PAM is an M-protein variant that displays high affinity binding to human plasminogen (hPg) (9,(11)(12)(13)(14). The secreted protein SK2b, from GAS subsequently activates PAM-bound hPg to hPm through cleavage of the R 561 V 562 peptide bond and removal of a N-terminal 77-residue activation peptide (6,11,(15)(16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

Group A Streptococcus-Induced Activation of Human Plasminogen Is Required for Keratinocyte Wound Retraction and Rapid Clot Dissolution

Zhong

Nguyen

et al. 2021

Front. Cardiovasc. Med.

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Invasive outcomes of Group A Streptococcus (GAS) infections that involve damage to skin and other tissues are initiated when these bacteria colonize and disseminate via an open wound to gain access to blood and deeper tissues. Two critical GAS virulence factors, Plasminogen-Associated M-Protein (PAM) and streptokinase (SK), work in concert to bind and activate host human plasminogen (hPg) in order to create a localized proteolytic environment that alters wound-site architecture. Using a wound scratch assay with immortalized epithelial cells, real-time live imaging (RTLI) was used to examine dynamic effects of hPg activation by a PAM-containing skin-trophic GAS isolate (AP53R+S−) during the course of infection. RTLI of these wound models revealed that retraction of the epithelial wound required both GAS and hPg. Isogenic AP53R+S− mutants lacking SK or PAM highly attenuated the time course of retraction of the keratinocyte wound. We also found that relocalization of integrin β1 from the membrane to the cytoplasm occurred during the wound retraction event. We devised a combined in situ-based cellular model of fibrin clot-in epithelial wound to visualize the progress of GAS pathogenesis by RTLI. Our findings showed GAS AP53R+S− hierarchically dissolved the fibrin clot prior to the retraction of keratinocyte monolayers at the leading edge of the wound. Overall, our studies reveal that localized activation of hPg by AP53R+S−via SK and PAM during infection plays a critical role in dissemination of bacteria at the wound site through both rapid dissolution of the fibrin clot and retraction of the keratinocyte wound layer.

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Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence

Cited by 7 publications

References 56 publications

Relationships Between Plasminogen-Binding M-Protein and Surface Enolase for Human Plasminogen Acquisition and Activation in Streptococcus pyogenes

Relationships Between Plasminogen-Binding M-Protein and Surface Enolase for Human Plasminogen Acquisition and Activation in Streptococcus pyogenes

Inactivation of the lysine binding sites of human plasminogen (hPg) reveals novel structural requirements for the tight hPg conformation, M-protein binding, and rapid activation

Group A Streptococcus-Induced Activation of Human Plasminogen Is Required for Keratinocyte Wound Retraction and Rapid Clot Dissolution

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